Therapeutic agents for alzheimer&#39;s disease and cancer

ABSTRACT

To provide a therapeutic drug for Alzheimer&#39;s disease and/or a cancer. The therapeutic drug for Alzheimer&#39;s disease and/or a cancer contains an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative.

TECHNICAL FIELD

The present invention relates to a therapeutic drug for Alzheimer'sdisease and/or a cancer, the drug containing an anti-nicastrin antibody.

BACKGROUND ART

In Japan, the three most common death-causing diseases are cancer(30.3%), cardiac disease (15.3%), and cerebrovascular disease (15.2%).As the population ages, the percentage of patients with such diseasesincreases, which greatly affects medical costs required for treatment ornursing care. In recent years, a nursing-care insurance system forcerebrovascular disease patients has been established as a nationalpolicy.

In Japan, the number of deaths from cancer was 320,315 (i.e., 253.9 per100,000) in 2004. In Japan, in 2003, lung cancer (22.3%) was rankedfirst among cancer deaths in men, followed by gastric cancer (17.2%) andliver cancer (12.5%), whereas colon cancer (14.6%) was ranked firstamong cancer deaths in women, followed by gastric cancer (14.2%) andlung cancer (12.3%). According to a report by the National Cancer Centerin Japan, regarding five-year survival rates for major types of cancer,the five-year survival rate of pancreatic cancer patients is the lowest(only a few percent), followed by that of patients with gallbladdercancer, lung and bronchial cancer, liver cancer, esophageal cancer, etc.Ohno, Nakamura, et al., have estimated that the number of new cases ofmale cancers will be 501,000 in 2020 (major sites of cancer: lung,prostate gland, stomach, colon, liver, etc.), whereas the number of newcases of female cancers will be 337,000 in 2020 (major sites of cancer:breast, colon, stomach, lung, uterus, rectum, liver, etc.). Thus, canceris predicted to become a major death-causing disease in future (as isthe case at present), and development of a therapy for cancer isessential.

Cancer therapy has changed with the times. Recently, in addition tohitherto performed surgery, drug therapy, and radiotherapy, endoscopicresection of cancer tissue has been carried out, and chemotherapy foroutpatients has been performed more and more. However, about 40% ofcancer cases are treated through surgery at present, and radiotherapy orchemotherapy is less effective for some cancers (e.g., pancreaticcancer). In some cancer cases, chemotherapy can reduce a size of cancertissue, but encounters difficulty in completely curing the disease. Inmany refractory cancer cases, adverse reactions to an anticancer agent(i.e., side effects thereof) are more pronounced than the effects of thedrug.

Cerebrovascular diseases are classified into a cognitive disorder, whichis caused by vascular disorder, and Alzheimer's disease, which is aneurodegenerative disease. In Japan, a number of patients with dementiacaused by Alzheimer's disease (AD) has increased with adoption ofEuropeanized and Americanized meals and aging of the population.

AD is a neurodegenerative disease which develops various intellectualdysfunctions (including memory impairment) due to degeneration or lossof cerebral cortical neurons. An AD brain is characterized byaccumulation of an abnormal protein called “β-amyloid,” which is closelyrelated to loss of neurons (Non-Patent Document 1).

β-Amyloid is accumulated in an AD brain in the pathological form ofsenile plaque or vascular amyloid. From the biochemical viewpoint,β-amyloid is formed of Aβ peptide including 40 to 42 amino acidresidues. Aβ is produced from APP (amyloid precursor protein) throughtwo-step cleavage and is secreted extracellularly. In the second step, aC-terminal fragment of APP is cleaved at an intramembrane site by theprotease activity of the enzyme γ-secretase, and the thus-formed Aβ isreleased extracellularly. Cleavage of the C-terminal fragment of APPoccurs at different sites; i.e., at position 40 (90%) and at position 42(10%) (Non-Patent Document 2). Aβ42 is more highly aggregated in theform of β-amyloid and is preferentially accumulated in an AD brain froman early stage (Non-Patent Document 3).

As has been shown, presenilin (PS) protein, which is an expressionproduct of a major pathogenic gene of familial AD, corresponds to acatalytic subunit of γ-secretase, which is a membrane-associatedaspartic protease (Non-Patent Documents 4 and 5).

γ-Secretase has been shown to be involved not only in AD but also inNotch signaling (Non-Patent Document 6). As has been known, aγ-secretase inhibitor (i.e., a low-molecular-weight compound) inducesapoptosis in Kaposi's sarcoma (Non-Patent Document 7) or inhibitssurvival of T-ALL cells (Non-Patent Document 8). However, it has beenreported that a γ-secretase inhibitor may promote malignanttransformation (Non-Patent Document 9). Thus, inhibition of Notchsignaling does not necessarily induce cell death in all cancers, and inthe future studies will be carried out to determine whether or not aγ-secretase inhibitor can be used as a therapeutic drug for cancer.

Under such circumstances, γ-secretase has been considered important as atherapeutic target for AD or cancer, but a cancer therapeutic drug basedon γ-secretase has not successfully been developed for, for example, thefollowing reason. Since γ-secretase is a complex formed of a pluralityof membrane proteins and exhibits protease activity in the membrane,difficulty is encountered in reconstituting γ-secretase whilemaintaining protease activity, and drug screening is not properlycarried out by use of γ-secretase.

As has been known, human active γ-secretase complex is a large membraneprotein complex having a molecular weight of 250 to 500 kDa or more andincluding the following four proteins: presenilin, nicastrin (NCT),APH-1, and PEN-2. That is, nicastrin is a constituent molecule ofγ-secretase. Many attempts have been made to search for γ-secretaseactivity inhibitors by use of low-molecular-weight compounds, but noreport has been provided to show a result of an experiment by use of ananti-nicastrin antibody for development of a γ-secretase activityinhibitor or a therapy for AD and/or cancer. Although there are many ADand cancer patients, a good drug for a treatment of the diseases has notyet been provided. Development of a therapeutic drug for AD or cancercould reduce burden of nursing care as a matter of course, along withmedical costs.

-   Non-Patent Document 1: Selkoe D J., Physiol. Rev. 2001, 81 (2):    741-766, Alzheimer's disease: genes, proteins, and therapy-   Non-Patent Document 2: Suzuki N., et al. Science 264: 1336, 1994-   Non-Patent Document 3: Iwatsubo T., Odaka A., Suzuki N., Mizusawa    H., Nukina N., Ihara Y., Neuron. 1994, 13 (1): 45-53-   Non-Patent Document 4: Wolfe M S., Xia W., Ostaszewski B L., Diehl T    S., Kimberly W T., Selkoe D J. (1999), Nature 398 (6727): 513-517-   Non-Patent Document 5: Li Y M., Xu M., Lai M T., Huang Q., Castro J    L., DiMuzio Mower J., Harrison T., Lellis C., Nadin A., Neduvelil J    G., Register R B., Sardana M K., Shearman M S., Smith A L., Shi X    P., Yin K C., Shafer J A., Gardell S J. (2000), Nature 2000 Jun. 8,    405 (6787): 689-94-   Non-Patent Document 6: J. Biol. Chem. 2001 Aug. 10; 276 (32):    30018-30023-   Non-Patent Document 7: Oncogene. 2005 Sep. 22; 24 (42): 6333-6344-   Non-Patent Document 8: Mol. Cell. Biol. 2003 January; 23 (2):    655-664-   Non-Patent Document 9: Br. J. Cancer. 2005 Sep. 19; 93 (6): 709-718

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention provides a new therapeutic drug for AD or acancer. The present invention also provides a screening method forselecting such a therapeutic drug.

Means for Solving the Problems

The present inventors have succeeded in expressing active γ-secretase byuse of budding baculovirus (see WO 2005/038023). The present inventorshave screened anti-nicastrin antibodies on a basis of active γ-secretaseactivity by use of budding baculovirus, and as a result have found thatan excellent anti-nicastrin antibody is useful as a therapeutic drug forAD and/or a cancer, since the anti-nicastrin antibody exhibitsγ-secretase-neutralizing activity and inhibits proliferation ofNotch-expressing cells and/or improves survival rate. The presentinvention has been accomplished on the basis of this finding. Also, thepresent inventors have found that an anti-nicastrin antibody inhibitsreaction between nicastrin and a γ-secretase substrate (i.e., apolypeptide including the intramembrane sequence of a receptor or APP),and thus this reaction system can be employed for selecting, throughscreening, an antibody which inhibits γ-secretase activity. The presentinvention has been accomplished also on the basis of this finding.

Accordingly, the present invention provides a therapeutic drug for ADand/or a cancer containing an anti-nicastrin antibody, a derivative ofthe antibody, or a fragment of the antibody or the derivative.

The present invention also provides a screening method for selecting anantibody which inhibits γ-secretase activity, characterized bycomprising reacting nicastrin with a γ-secretase substrate in a presenceof a test antibody.

The present invention also provides use of an anti-nicastrin antibody, aderivative of the antibody, or a fragment of the antibody or thederivative for producing a therapeutic drug for Alzheimer's diseaseand/or a cancer.

The present invention also provides a method for treatment ofAlzheimer's disease and/or a cancer, characterized by comprisingadministering an anti-nicastrin antibody, a derivative of the antibody,or a fragment of the antibody or the derivative to a subject in needthereof.

Effects of the Invention

According to the therapeutic drug for AD and/or a cancer of the presentinvention, γ-secretase activity can be inhibited by an anti-nicastrinantibody, to thereby treat Alzheimer's disease and/or a cancer.

According to the screening method of the present invention, the reactionsystem between nicastrin and a γ-secretase substrate can be employed forselecting an antibody which inhibits γ-secretase activity; i.e., anantibody effective for the treatment of Alzheimer's disease and/or acancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of screening of anti-nicastrin antibodies throughBV-ELISA.

FIG. 2 shows results of screening of anti-nicastrin antibodies throughwestern blot analysis by use of BV.

FIG. 3 shows results of western blot analysis of various forms ofnicastrin expressed in COS-7 cells. FIG. 3 discloses the “DYIGS” peptideas SEQ ID NO: 19.

FIG. 4 shows results of an experiment for evaluating cross-reactivity ofanti-nicastrin antibodies to deglycosylated nicastrin (“O” representsEndo H-resistant nicastrin; “black dot” represents completelydeglycosylated nicastrin; and “Δ” represents neuraminidase-desialylatednicastrin).

FIG. 5 shows results of IP of nicastrin from a soluble membrane fractionof HeLa cell by use of anti-nicastrin antibodies.

FIG. 6 shows results of treatment of a HeLa cell lysate with trypsin.

FIG. 7 shows results of immunostaining of HeLa cells by use ofanti-nicastrin antibodies.

FIG. 8 shows results of immunostaining of NKO cells and NKO/NCT cells byuse of anti-nicastrin antibodies.

FIG. 9 shows results of co-staining by use of anti-nicastrin antibodiesand antibodies to various marker proteins.

FIG. 10 shows results of co-staining by use of anti-nicastrin antibodiesand cholera toxin subunit B (CTB).

FIG. 11 shows an effect of anti-nicastrin antibodies on in vitroγ-secretase activity.

FIG. 12 shows an effect of DAPT on a viability of HeLa cells or A549cells.

FIG. 13 shows an effect of inhibition of nicastrin expression on aviability of A549 cells.

FIG. 14 shows results of inhibition of expression of endogenousnicastrin in A549 cells by siRNA.

FIG. 15 shows an effect of anti-nicastrin antibodies on the viability ofA549 cells.

FIG. 16 shows mutation sites of Notch1 gene in various T-ALL-derivedcells.

FIG. 17 shows results of western blot analysis of Notch1 gene productsin various T-ALL-derived cell lysates.

FIG. 18 shows an effect of a γ-secretase inhibitor YO on proliferationof various T-ALL-derived cells.

FIG. 19 shows an effect of an anti-nicastrin antibody on proliferationof DND-41 cells.

FIG. 20 shows results of western blot analysis of anti-nicastrinantibodies and a nicastrin-N100 fraction.

FIG. 21 shows an effect of anti-nicastrin antibodies on inhibition ofbinding between nicastrin and N100-FLAG.

FIG. 22A shows an effect of an anti-nicastrin antibody on inhibition ofγ-secretase activity in living cells.

FIG. 22B shows an effect of an anti-nicastrin antibody on inhibition ofγ-secretase activity in living cells.

FIG. 22C shows an effect of an anti-nicastrin antibody on inhibition ofγ-secretase activity in living cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will next be described in detail.

The present invention is directed to a therapeutic drug for AD and/or acancer containing an anti-nicastrin antibody, a derivative of theantibody, or a fragment of the antibody or the derivative; to use of theantibody, the derivative, or the fragment for producing such atherapeutic drug; and to a method for the treatment of Alzheimer'sdisease and/or a cancer.

In the present invention, as described hereinbelow, the anti-nicastrinantibody derivative encompasses a modified anti-nicastrin antibody andan anti-nicastrin antibody to which a compound exhibiting a desiredpharmaceutical activity has been bound.

Nicastrin is a membrane protein and forms a complex to exhibit.gamma.-secretase activity. An amino acid sequence of nicastrin and asequence of the gene coding therefor are disclosed in GenBank™ number(NM_(—)015331) (SEQ ID Nos: 1 and 2). As used herein, “nicastrinprotein” encompasses both full-length nicastrin protein and a fragmentof nicastrin. As used herein, “fragment of nicastrin” refers to apolypeptide which includes a predetermined region of nicastrin proteinand which does not necessarily have a function of natural nicastrinprotein.

Nicastrin protein, which is employed as an antigen in the presentinvention, is preferably human nicastrin protein, but is not necessarilylimited thereto. The nicastrin protein employed in the present inventionmay be nicastrin derived from any non-human species, such as caninenicastrin, feline nicastrin, mouse nicastrin, hamster nicastrin, ordrosophila nicastrin. Preferably, an antibody selected by use ofnicastrin protein neutralizes human active γ-secretase includingnicastrin as a constituent molecule.

Human active γ-secretase is a large-molecule membrane protein complexhaving a molecular weight of 250 to 500 kDa or more and including thefollowing four proteins: fragmented presenilin, nicastrin, APH-1, andPEN-2.

The active γ-secretase employed in the present invention may be preparedthrough any of methods described in the Examples hereinbelow (WO2005/038023). Natural human active γ-secretase may be prepared from ahuman brain homogenate, but is very difficult to employ for screening ofγ-secretase inhibitors. Therefore, the active γ-secretase employed ispreferably prepared through the method by the present inventors forsuccessfully expressing an active γ-secretase complex by use of buddingbaculovirus (see WO 2005/038023).

In the present invention, γ-secretase activity is determined through amethod by Yasuko Takahashi, et al. (J. Biol. Chem. 2003 May 16; 278(20): 18664-70), which is a generally known method. Specifically, a testsubstance is mixed with microsomes (serving as an enzyme) prepared frombrain tissue or cultured cells, and the mixture is incubated at 4° C.for 12 hours. Subsequently, 1 μM C100FmH serving as a substrate is addedto the reaction mixture, and the mixture is incubated at 37° C. for 12hours. Thereafter, an amount of amyloid-β is measured through sandwichELISA, to thereby determine γ-secretase activity.

Preparation of Anti-Nicastrin Antibody

Preferably, the anti-nicastrin antibody employed in the presentinvention not only binds specifically to nicastrin protein, but alsoneutralizes human active γ-secretase. No particular limitation isimposed on an origin, type (monoclonal or polyclonal), and form of theanti-nicastrin antibody. Specifically, the anti-nicastrin antibody maybe a known antibody such as a mouse antibody, a rat antibody, an avianantibody, a human antibody, a chimera antibody, and a humanized(CDR-grafted) antibody. The anti-nicastrin antibody is preferably ahuman, chimera, or humanized monoclonal antibody.

Examples of the anti-nicastrin monoclonal antibody include a monoclonalantibody produced by a hybridoma, and a monoclonal antibody produced ina host transformed with an expression vector containing a gene for theantibody through a genetic engineering technique.

Basically, a hybridoma which produces the monoclonal antibody may beprepared through a known technique as described below. Specifically, thehybridoma may be prepared through the following procedure: a mammal isimmunized with nicastrin protein serving as a sensitizing antigenthrough a customary immunization method; the resultant immunocyte isfused with a known parental cell through a customary cell fusion method;and a cell for producing the monoclonal antibody is selected through acustomary screening method.

Specifically, the monoclonal antibody can be prepared as follows.

Firstly, nicastrin protein, which is employed as a sensitizing antigenfor preparing the monoclonal antibody, is obtained through expression ofa nicastrin gene/amino acid sequence disclosed in GenBank number(NM_(—)015331). Specifically, an appropriate host cell is transformedwith a known expression vector system containing the gene sequenceencoding nicastrin, and then human nicastrin protein of interest ispurified from the resultant host cell or a culture supernatant of thecell through a known method. Alternatively, natural nicastrin proteinmay be employed after being purified.

Subsequently, the thus-purified nicastrin protein is employed as asensitizing antigen. Alternatively, a partial peptide of the nicastrinprotein may be employed as a sensitizing antigen. Such a partial peptidemay be obtained through chemical synthesis on the basis of the aminoacid sequence of nicastrin protein, through integration of a portion ofthe nicastrin gene into an expression vector, or through degradation ofnatural nicastrin protein by use of protease. No particular limitationis imposed on a site or size of a nicastrin protein portion employed asa partial peptide.

No particular limitation is imposed on the mammal which is immunizedwith the sensitizing antigen, but preferably, the mammal is selected inconsideration of compatibility of the resultant immunocyte with aparental cell employed for cell fusion. In general, a rodent (e.g.,mouse, rat, or hamster), avian, rabbit, monkey, or the like is employed.

Immunization of an animal with the sensitizing antigen is carried outthrough a known method. For example, in a generally employedimmunization method, the sensitizing antigen is intraperitoneally orsubcutaneously injected into a mammal. Specifically, the sensitizingantigen is diluted by PBS (phosphate-buffered saline), saline, or thelike, to form a suspension of an appropriate volume. If desired, theresultant suspension is mixed with an appropriate volume of a commonadjuvant (e.g., Freund's complete adjuvant). After emulsification of theresultant mixture, the emulsion is administered to a mammal severaltimes every 4 to 21 days. Upon immunization with the sensitizingantigen, an appropriate carrier may be employed. Particularly when thesensitizing antigen is a partial peptide of low molecular weight,preferably, the partial peptide employed for immunization is bound to acarrier protein such as albumin or keyhole limpet hemocyanin.

After immunization of a mammal as described above, and followingconfirmation of an increase in serum level of an antibody of interest,immunocytes are collected from the mammal and then subjected to cellfusion. The type of immunocytes is particularly preferably splenocyte.

A mammalian myeloma cell is employed as a parental cell which is fusedwith the aforementioned immunocyte. The myeloma cell employed ispreferably a known cell line; for example, P3 (P3x63Ag8.653) (J.Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics inMicrobiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. andMilstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H., et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M., et al., Nature(1978) 276, 269-270), FO (de St. Groth, S. F., et al., J. Immunol.Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978)148, 313-323), or R210 (Galfre, G., et al., Nature (1979) 277, 131-133).

Cell fusion between the aforementioned immunocyte and myeloma cell maybe basically carried out through a known method, such as a method ofKohler, Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol.(1981) 73, 3-46).

More specifically, the aforementioned cell fusion is carried out in acommon nutrient culture medium in the presence of, for example, a cellfusion promoter. Examples of the cell fusion promoter employed includepolyethylene glycol (PEG) and Sendai virus (HVJ). If desired, anauxiliary agent (e.g., dimethyl sulfoxide) may be further added in orderto enhance cell fusion efficiency.

The ratio of the immunocyte and myeloma cell employed may be determinedas desired. For example, an amount of the immunocyte is preferably 1 to10 times that of the myeloma cell. Examples of the culture medium whichmay be employed for the aforementioned cell fusion include RPMI 1640medium and MEM medium, which are suitable for proliferation of theaforementioned myeloma cell line; and culture media which are generallyemployed for such a cell culture. Such a culture medium may be employedin combination with a serum supplement such as fetal calf serum (FCS).

In the cell fusion, predetermined amounts of the aforementionedimmunocyte and myeloma cell are well-mixed in any of the aforementionedculture media, and a solution of PEG (e.g., PEG having an averagemolecular weight of about 1,000 to about 6,000) which has been heated inadvance to about 37° C. is added to the resultant mixture in apredetermined amount (generally 30 to 60% (w/v)), followed by mixing, tothereby yield a hybridoma of interest. Subsequently, a procedureincluding sequential addition of an appropriate culture medium andremoval of a supernatant obtained through centrifugation is repeated, tothereby remove substances (e.g., a cell fusion promoter) which are notsuitable for growth of the hybridoma.

Separation of the thus-yielded hybridoma is carried out throughculturing in a common selective culture medium such as a HAT medium (amedium containing hypoxanthine, aminopterin, and thymidine). A culturingin the aforementioned HAT medium is continued for a sufficient period oftime (generally several days to several weeks) for apoptosis of cells(i.e., non-fused cells) other than the hybridoma of interest.Subsequently, a customary limiting dilution technique is performed forscreening and monocloning of the hybridoma which produces a targetantibody.

An antibody which recognizes nicastrin protein may be prepared through amethod described in WO 03/104453.

Screening and monocloning of a target antibody may be carried outthrough a known screening method on a basis of antigen-antibodyreaction. For example, an antigen is bound to a carrier (e.g., beadsmade of polystyrene or a similar material, or a commercially available96-well microtiter plate) and then reacted with a culture supernatant ofthe hybridoma, and subsequently the carrier is washed, followed byreaction with, for example, an enzyme-labeled secondary antibody, tothereby determine whether or not the culture supernatant contains atarget antibody which reacts with a sensitizing antigen. Cloning of thehybridoma which produces a target antibody may be performed through, forexample, a limiting dilution technique. In this case, the antigen may bean antigen employed in immunization.

In addition to preparation of the aforementioned hybridoma throughimmunization of a non-human animal with an antigen, a human antibody ofinterest having binding activity to nicastrin may be prepared bysensitizing human lymphocyte with nicastrin in vitro, and fusing thethus-sensitized lymphocyte with a human-derived myeloma cell havingpermanent division capacity (see JP-A-01-059878). Alternatively,nicastrin serving as an antigen may be administered to a transgenicanimal having all of the human antibody gene repertories, to therebyyield a cell which produces an anti-nicastrin antibody, and a humanantibody against nicastrin may be obtained from the cell after it hasbeen immortalized (see WO 94/25585, WO 93/12227, WO 92/03918, and WO94/02602).

The thus-prepared monoclonal-antibody-producing hybridoma can besubcultured in a common culture medium and can be stored in liquidnitrogen for a long period of time.

A monoclonal antibody is produced from the hybridoma through, forexample, a method in which the hybridoma is cultured by a customarytechnique, and the monoclonal antibody is obtained from the resultantculture supernatant; or a method in which the hybridoma is administeredto a mammal exhibiting compatibility with the hybridoma to therebyproliferate the hybridoma, and the monoclonal antibody is obtained fromascitic fluid of the mammal. The former method is suitable for obtaininga monoclonal antibody of high purity, whereas the latter method issuitable for a mass production of a monoclonal antibody.

The monoclonal antibody employed in the present invention may be arecombinant antibody. Such a recombinant antibody is produced throughthe following procedure: the antibody gene is cloned from the hybridoma;the gene is integrated into an appropriate vector; and the vector isintroduced into a host, followed by production of the recombinantantibody through a genetic recombination technique (see, for example,Vandamme, A. M., et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).

Specifically, mRNA encoding a variable (V) region of an anti-nicastrinantibody is isolated from the hybridoma which produces theanti-nicastrin antibody. Isolation of mRNA is carried out as follows.Total RNA is prepared through a known method such as the guanidineultracentrifugation method (Chirgwin, J. M., et al., Biochemistry (1979)18, 5294-5299) or the AGPC method (Chomczynski, P., et al., Anal.Biochem. (1987) 162, 156-159), and target mRNA is prepared by means of,for example, mRNA Purification Kit (product of Pharmacia).Alternatively, mRNA may be directly prepared by means of QuickPrep mRNAPurification Kit (product of Pharmacia).

The thus-obtained mRNA is employed for synthesis of cDNA of the antibodyV region by use of reverse transcriptase. Synthesis of cDNA is carriedout by means of, for example, AMV Reverse Transcriptase First-strandcDNA Synthesis Kit (product of Seikagaku Corporation). Alternatively,synthesis and amplification of cDNA may be carried out by means of, forexample, 5′-Ampli FINDER RACE Kit (product of Clontech) or the 5′-RACEmethod using PCR (Frohman, M. A., et al., Proc. Natl. Acad. Sci. USA(1988) 85, 8998-9002; Belyavsky, A., et al., Nucleic Acids Res. (1989)17, 2919-2932).

A target DNA fragment is purified from the resultant PCR product andligated to vector DNA. Subsequently, a recombinant vector is preparedfrom the vector DNA and then introduced into Escherichia coli or thelike, followed by colony selection, to thereby prepare a recombinantvector of interest. The nucleotide sequence of the target DNA fragmentis determined through a known method such as a dideoxynucleotide chaintermination method.

DNA encoding the V regions of a target anti-nicastrin antibody isobtained, and then the DNA is integrated into an expression vectorcontaining DNA encoding constant regions (C regions) of the targetantibody.

In order to produce the anti-nicastrin antibody employed in the presentinvention, the gene for the antibody is integrated into an expressionvector so that the gene can be expressed under a control of anexpression regulatory region (e.g., an enhancer or a promoter).Subsequently, a host cell is transformed with this expression vector forexpression of the antibody.

The gene for the antibody may be expressed by transforming a host cellwith both an expression vector containing the DNA encoding a heavy chain(H chain) of the antibody and an expression vector containing the DNAencoding a light chain (L chain) of the antibody, or by transforming ahost cell with a single expression vector containing the DNA encodingthe heavy and light chains of the antibody (see WO 94/11523).

In addition to the aforementioned host cell, a transgenic animal may beemployed for production of a recombinant antibody. For example, anantibody gene is inserted into a gene encoding a protein producedspecifically in milk (such as goat β-casein) to prepare a fusion gene. ADNA fragment including the fusion gene having the inserted antibody geneis injected into an embryo of a goat, and this embryo is implanted intoa female goat. An antibody of interest is obtained from milk produced bytransgenic goats born from the goat impregnated with the embryo orprogeny thereof. In order to increase an amount of theantibody-containing milk produced by the transgenic goats, hormones maybe administered to the transgenic goats as appropriate (Ebert, K. M. etal., Bio/Technology (1994) 12, 699-702).

In the present invention, in addition to the aforementioned antibodies,an artificially modified, genetically recombinant antibody (e.g., achimera antibody or a humanized antibody) may be employed. Such amodified antibody may be produced through a known method.

Specifically, a chimera antibody is prepared through the followingprocedure: the above-obtained DNA encoding the antibody V regions isligated to the DNA encoding the human antibody C regions; thethus-ligated DNA is integrated into an expression vector; and theexpression vector is introduced into a host for production of thechimera antibody. Through this known procedure, a chimera antibodyuseful for the present invention can be prepared.

A humanized antibody is also called a “reshaped human antibody” and isobtained by grafting a complementarity-determining regions (CDRs) of anantibody from a non-human mammal (e.g., mouse) into thecomplementarity-determining regions of a human antibody. Typical generecombination techniques for preparing such a humanized antibody areknown (see European Patent Application Laid-Open (EP) No. 125023 and WO96/02576).

Specifically, a DNA sequence designed to ligate a CDRs of a mouseantibody to a framework regions (FRs) of a human antibody is synthesizedthrough PCR employing, as primers, several oligonucleotides prepared tohave portions overlapping terminal regions of both the CDRs and FRs (seethe method described in WO 98/13388).

The framework regions of the human antibody ligated via the CDRs areselected in such a manner that the complementarity-determining regionsform a proper antigen-binding site. If necessary, amino acid residues inthe framework regions of the antibody variable regions may besubstituted so that the complementarity-determining regions of areshaped human antibody form a proper antigen-binding site (Sato, K., etal., Cancer Res. (1993) 53, 851-856).

The C regions employed in a chimera antibody or a humanized antibody maybe those of a human antibody; for example, Cγ1, Cγ2, Cγ3, and Cγ4 in theH chain, and Cκ and Cλ in the L chain. The C regions of the humanantibody may be modified so as to improve a stability of the antibody orto achieve stable production thereof.

The chimera antibody includes the variable regions of an antibodyderived from a non-human mammal and the constant regions of a humanantibody. Meanwhile, the humanized antibody includes thecomplementarity-determining regions of an antibody derived from anon-human mammal and the framework regions and C regions of a humanantibody. The humanized antibody is useful as an active ingredient of atherapeutic agent, since it exhibits low antigenicity in a human body.

The anti-nicastrin antibody employed in the present invention is notlimited to the whole antibody molecule. So long as the anti-nicastrinantibody binds to nicastrin protein, the antibody may be an antibodyfragment, derivatives of the antibody (including a modified antibody,and an antibody bound to a compound exhibiting a desired pharmaceuticalactivity), a divalent antibody, or a monovalent antibody. Theanti-nicastrin antibody is preferably an antibody which neutralizeshuman active γ-secretase.

Examples of the antibody fragment include Fab, F(ab′)₂, Fab/c having Fabhaving one Fv and complete Fc, and single-chain Fv (scFv) in which Fvfragments of the H or L chain are linked together with an appropriatelinker. Specifically, an antibody is treated with an enzyme (e.g.,papain or pepsin) to produce an antibody fragment. Alternatively, a geneencoding such an antibody fragment is constructed and introduced into anexpression vector, followed by expression in an appropriate host cell(see, for example, Co, M. S., et al., J. Immunol. (1994) 152, 2968-2976;Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496,Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology(1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods inEnzymology (1989) 121, 652-663; Rousseaux, J., et. al., Methods inEnzymology (1989) 121, 663-669; and Bird, R. E., et al., TIBTECH (1991)9, 132-137).

A single-chain Fv (scFv) is obtained by linking the H chain V region andL chain V region of an antibody. In the scFv fragment, the H chain Vregion and the L chain V region are linked by a linker (preferably, apeptide linker) (Huston, J. S., et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883). The H chain V region and the L chain V region inthe scFv fragment may be derived from any of the antibodies describedherein. The peptide linker employed for linking the V regions is, forexample, any single-stranded peptide including 12 to 19 amino acidresidues.

DNA encoding the scFv fragment is obtained through PCR amplificationemploying, as a template, an entire sequence of the DNA encoding the Hchain or H chain V region of the aforementioned antibody or the DNAencoding the L chain or L chain V region of the antibody, or a portionof the DNA sequence encoding an amino acid sequence of interest, incombination with a primer pair defining both ends of the DNA sequence,followed by amplification employing the DNA encoding a peptide linkerregion in combination with a primer pair which defines both ends of theDNA so that the respective ends are linked to the H and L chains.

Once the DNA encoding the scFv fragment is prepared, an expressionvector containing the DNA and a host transformed with the expressionvector can be obtained through a customary method, and the scFv fragmentcan be obtained through a customary method by use of the host.

Such an antibody fragment may be produced by a host after a gene for thefragment has been obtained and expressed in a manner similar to thatdescribed above. As used herein, the term “antibody” also encompassessuch an antibody fragment.

Also, a modified anti-nicastrin antibody prepared through conjugation ofa molecule (e.g., polyethylene glycol (PEG) or a sugar chain) to ananti-nicastrin antibody may be employed. Through such modification, ahalf-life of the anti-nicastrin antibody can be prolonged, andhydrolysis or elimination thereof can be reduced in blood. As usedherein, the term “antibody” also encompasses such a modified antibody.Such a modified antibody may be prepared through chemical modificationof the above-obtained antibody or a fragment thereof. Methods formodifying antibodies have already been established in the art.

Also, the antibody employed in the present invention may be a bispecificantibody. The bispecific antibody may have antigen-binding sitesrecognizing different epitopes of NCT molecule. A bispecific antibodymay be prepared by binding HL pairs of two antibodies, or may beobtained from a bispecific-antibody-producing fused cell preparedthrough fusion of hybridomas producing different monoclonal antibodies.Alternatively, a bispecific antibody may be prepared through a geneticengineering technique.

The above-constructed gene for the antibody may be expressed through aknown method, to thereby yield the antibody. In the case where amammalian cell is employed, the antibody gene may be expressed byfunctionally binding a common useful promoter, the gene which isexpressed, and a polyA signal downstream of a 3′-end thereof. Examplesof the promoter/enhancer which may be employed include humancytomegalovirus immediate early promoter/enhancer.

Other promoters/enhancers which may be employed for antibody expressionin the present invention include viral promoters/enhancers such asretrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40); andpromoters/enhancers derived from mammalian cells, such as humanelongation factor 1α (HEF1α).

When SV40 promoter/enhancer is employed, gene expression can be readilycarried out through a method of Mulligan, et al. (Nature (1979) 277,108), whereas when HEF1α promoter/enhancer is employed, gene expressioncan be readily carried out through a method of Mizushima, et al.(Nucleic Acids Res. (1990) 18, 5322).

In the case where Escherichia coli are employed, the gene for theantibody can be expressed by functionally binding a common usefulpromoter, a signal sequence for secreting the antibody, and the antibodygene which is expressed. Examples of the promoter which may be employedinclude lacZ promoter and araB promoter. When lacZ promoter is employed,the gene can be expressed through a method of Ward, et al. (Nature(1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), whereas when araBpromoter is employed, the gene can be expressed through a method ofBetter, et al. (Science (1988) 240, 1041-1043).

When the antibody is produced in a periplasm of Escherichia coli, a pe1Bsignal sequence (Lei, S. P., et al., J. Bacteriol. (1987) 169, 4379) maybe employed as a signal sequence for secreting the antibody. Theantibody produced in the periplasm is isolated and then employed byappropriately refolding a structure of the antibody.

Replication origins which may be employed include those derived fromSV40, polyomavirus, adenovirus, bovine papilloma virus (BPV). In orderto increase gene copy number in a host cell system, the expressionvector employed may contain a selective marker such as aminoglycosidetransferase (APH) gene, thymidine kinase (TK) gene, Escherichia colixanthine-guanine phosphoribosyl transferase (Ecogpt) gene, ordihydrofolate reductase (dhfr) gene.

Any expression system such as a eukaryotic or prokaryotic system may beused for production of the antibody employed in the present invention.Examples of the eukaryotic cell include animal cells of, for example,established mammalian cell line, cells of insect cell line, filamentousfungal cells, and yeast cells; and examples of the prokaryotic cellinclude cells of a bacterium such as Escherichia coli.

Preferably, the antibody employed in the present invention is expressedin a mammalian cell such as CHO, COS, myeloma, BHK, Vero, or HeLa cell.

Subsequently, the above-transformed host cell is cultured in vitro or invivo to produce a target antibody. Culturing of the host cell is carriedout through a known method. For example, DMEM, MEM, RPMI 1640, or IMDMmay be employed as a culture medium, and a serum supplement such asfetal calf serum (FCS) may be employed in combination.

The above-expressed or produced antibody can be isolated from cells or ahost animal and purified to homogeneity. Isolation and purification ofthe antibody employed in the present invention may be carried out bymeans of an affinity column. Examples of columns employing protein Acolumn include Hyper D, POROS, and Sepharose F.F. (products ofPharmacia). No particular limitation is imposed on the method forisolation/purification of the antibody, and the antibody may be isolatedor purified through any method which is generally employed forisolation/separation of proteins. For example, the antibody may beisolated/purified by appropriately selecting or combining chromatographycolumns other than the aforementioned affinity columns, filters,ultrafiltration, salting out, dialysis, etc. (Antibodies A LaboratoryManual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

As described in the Examples hereinbelow, the above-obtainedanti-nicastrin antibody recognizes nicastrin protein (i.e., aconstituent molecule of human active γ-secretase), binds specifically tonicastrin protein, and exhibits an activity to neutralize γ-secretase.In addition, the anti-nicastrin antibody has an ability to inhibitproliferation of γ-secretase-dependent cancer cells. Therefore, theanti-nicastrin antibody, a derivative of the antibody, or a fragment ofthe antibody or the derivative is effective as a therapeutic drug forAlzheimer's disease and/or a cancer.

Conceivably, the cancer which can be treated by the present invention isa nicastrin-expressing cancer and/or a γ-secretase-dependent cancer.

Examples of such a cancer include lung cancer and T-cell acutelymphoblastic leukemia.

As used herein, “nicastrin-expressing cancer” refers to a cancer inwhich nicastrin protein is produced through expression of the nicastringene; and “γ-secretase-dependent cancer” refers to a cancer in whichproliferation of cancer cells requires γ-secretase, and cancer cellproliferation is inhibited or cancer cells die through inhibition ofγ-secretase activity.

The anti-nicastrin antibody derivative or a fragment thereof alsoencompasses a product prepared by conjugating a compound exhibiting adesired pharmaceutical activity to the anti-nicastrin antibody or afragment thereof through a customary method. Such an anti-nicastrinantibody derivative may be employed in, for example, a missile therapyspecifically targeting nicastrin. As used herein, “compound exhibiting adesired pharmaceutical activity” refers to a compound exhibiting, forexample, a pharmaceutical activity to inhibit or promote a substance(e.g., an enzyme or a receptor) which directly or indirectly causessymptoms to progress.

Examples of compounds exhibiting a desired pharmaceutical activity forcancer treatment include a compound which causes damage to cancer cells,and a compound which provides or enhances cytotoxic activity (e.g., aradioisotope). The radioisotope employed may be any radioisotope knownto those skilled in the art, but is preferably ¹³¹I, ^(99m)Tc, ¹¹¹In, or⁹⁰Y.

Cancer treatment employing an antibody bound to aradioisotope-containing compound may be carried out through a methodknown to those skilled in the art. Specifically, firstly, a small amountof an antibody bound to a radioisotope-containing compound isadministered to a patient, followed by whole-body scintigraphy. Afterdetermination that a degree of binding between the antibody and normaltissue cells is low but the degree of binding between the antibody andcancer cells is high, a large amount of the radioisotope-bound antibodyis administered to the patient.

The therapeutic drug of the present invention may be prepared into adrug product by subjecting both the drug and a pharmaceuticallyacceptable carrier well known in the art to a drug preparation processsuch as mixing, dissolution, granulation, tableting, emulsification,encapsulation, or lyophilization.

For oral administration, the therapeutic drug of the present inventionmay be mixed with, for example, a pharmaceutically acceptable solvent,excipient, binder, stabilizer, or dispersant, and the mixture may beprepared into a dosage form such as tablet, pill, sugar-coated agent,soft capsule, hard capsule, solution, suspension, emulsion, gel, syrup,or slurry.

For parenteral administration, the therapeutic drug of the presentinvention may be mixed with, for example, a pharmaceutically acceptablesolvent, excipient, binder, stabilizer, or dispersant, and the mixturemay be prepared into a dosage form such as injection solution,suspension, emulsion, cream, ointment, inhalant, or suppository. Forformulation of an injection, the therapeutic drug of the presentinvention may be dissolved in an aqueous solution, preferably, aphysiologically compatible buffer (e.g., Hanks' solution, Ringersolution, or saline buffer). The composition may be in the form ofsuspension, solution, or emulsion in an oily or aqueous vehicle.Alternatively, the therapeutic drug may be produced in the form ofpowder, and, before use, the drug may be prepared into an aqueoussolution or suspension with, for example, sterile water. For inhalationadministration, the therapeutic drug of the present invention may bepowdered and may be prepared into a powder mixture together with anappropriate base such as lactose or starch. For production of asuppository, the therapeutic drug of the present invention may be mixedwith a conventional suppository base such as cocoa butter. Thetherapeutic drug of the present invention may be formulated into asustained-release drug product by encapsulating the drug in, forexample, a polymer matrix.

A dose of the therapeutic drug of the present invention or a number ofdoses thereof varies depending on a dosage form or administration routethereof, or the symptom, age, or body weight of a patient in needthereof. The therapeutic drug can be administered once to several timesper day so that a daily dose of the drug is generally about 0.001 mg toabout 1,000 mg per kg body weight, preferably about 0.01 mg to about 10mg per kg body weight.

Generally, the therapeutic drug is administered through a parenteralroute; for example, injection (e.g., subcutaneous injection, intravenousinjection, intramuscular injection, or intraperitoneal injection), ortransdermal, transmucosal, transnasal, or transpulmonary administration.However, no particular limitation is imposed on the administration routeof the therapeutic drug, and the drug may be orally administered.

A screening method for selecting an antibody which inhibits γ-secretaseactivity.

As described in the Examples hereinbelow, an anti-nicastrin antibody hasbeen found to inhibit reaction between nicastrin and a γ-secretasesubstrate (e.g., C99 or N99).

Therefore, the screening method of the present invention for selectingan antibody which inhibits γ-secretase activity is a promising methodfor searching a therapeutic drug for AD or cancer.

In the screening method of the present invention, nicastrin is reactedwith a γ-secretase substrate (e.g., a polypeptide formed of the entiretyor a portion of Notch receptor and/or APP (including the intramembranesequence)) in the presence of a test antibody, and the reaction betweennicastrin and the substrate is detected.

Specifically, nicastrin is reacted with a polypeptide formed of theentirety or a portion of the sequence of Notch or APP in the presence ofa test antibody through addition thereof, and whether or not the addedtest antibody inhibits the reaction is determined through a knowndetection method.

Alternatively, a test antibody is exposed to cells expressing nicastrinand a polypeptide formed of the entirety or a portion of the sequence ofNotch and/or APP, and whether or not a product is produced through thereaction between nicastrin and the polypeptide is determined through aknown detection method.

Preferably, the latter screening method is carried out. The latterscreening method requires a simpler screening process. In addition, whenthe latter screening method is carried out in combination with a knowndetection method, numerous test antibodies can be screened to determinewhether or not they inhibit γ-secretase activity within a short periodof time. Thus, a therapeutic drug for AD or cancer can be developedwithin a short period.

The γ-secretase substrate employed in the screening method may be apolypeptide formed of the entirety or a portion of Notch receptor(NM_(—)008714) (SEQ ID NO: 3) (including the intramembrane sequence)and/or a polypeptide formed of the entirety or a portion of APP protein(NM_(—)000484) (SEQ ID NO: 4) (including the intramembrane sequence).The polypeptide formed of the entirety or a portion of Notch receptor orAPP protein may be prepared through expression of a gene having asequence(5′-cacctcatgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtgctgctg-3′) (SEQ ID NO: 6) and encoding a polypeptide including theintramembrane sequence of Notch receptor(NH₂-HLMYVAAAAFVLLFFVGCGVLL-COOH) (SEQ ID NO: 5), or a gene having asequence(5′-ggtgcaatcattggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcaccttggtgatgctg-3′) (SEQ ID NO: 8) and encoding a polypeptide including theintramembrane sequence of APP protein(NH₂-GAIIGLMVGGVVIATVIVITLVML-COOH) (SEQ ID NO: 7). Particularlypreferably, the polypeptide formed of the entirety or a portion of Notchreceptor or APP protein is prepared through expression of a gene havinga sequence (SEQ ID NO: 10) and encoding 99 amino acid residues (No. 1711to No. 1809) of a protein of Notch receptor including the intramembranesequence(NH₂-VKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) (SEQ ID NO: 9) (the 99amino acid residues may be called “N99”), or a gene having a sequence(SEQ ID NO: 12) and encoding 99 amino acid residues (the C-terminus toNo. 99) of a protein of APP including the intramembrane sequence(NH₂-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN-COOH) (SEQ ID NO: 11) (the 99amino acid residues may be called “C99”).

The polypeptide serving as a γ-secretase substrate, which is formed ofthe entirety or a portion of a protein, is obtained from thecorresponding amino acid sequence or expressing a gene encoding theamino acid residues. Alternatively, the polypeptide is obtained from anatural product.

The polypeptide serving as a γ-secretase substrate, which is formed ofthe entirety or a portion of a protein, is preferably derived fromhuman. However, the origin of the polypeptide is not limited to human,and the polypeptide may be derived from any non-human species such asdog, cat, mouse, hamster, or drosophila.

The amino acid sequence of nicastrin or a γ-secretase substrate or thesequence of the gene coding therefor may be provided, before expressionthereof, with a tag sequence (e.g., V5 or FLAG sequence), which isselected in consideration of a detection method employed.

Whether or not a test antibody inhibits γ-secretase activity may bedetermined through a known technique such as co-immunoprecipitation(IP), western blotting, ELISA, reporter gene assay, a SPA beads method,a fluorescence polarization method, or a homogeneous time-resolvedfluorescence method. These techniques may be employed singly or incombination as appropriate.

For example, co-immunoprecipitation (IP) and western blotting may beemployed in combination. In this case, the amino acid sequence ofnicastrin or a peptide formed of the entirety or a portion of Notchreceptor or APP (including the intramembrane sequence) or the sequenceof the gene coding therefor is provided with a tag sequence (e.g., FLAGor V5 sequence) through a known method, and the protein or peptide isexpressed in a host cell.

Nicastrin or the peptide formed of the entirety or a portion of Notchreceptor or APP (including the intramembrane sequence) is extracted fromthe host cell by a known extraction method including lysis of the cellmembrane, followed by purification as appropriate.

The thus-extracted nicastrin is diluted with a culture medium and thenmixed with a test antibody, and reaction is carried out at 4° C. for 8to 12 hours. Thereafter, the Notch or APP peptide is added to thereaction mixture, followed by further mixing for three to four hours. AHEPES buffer containing 0.5% CHAPSO is employed as a buffer solution.

An antibody corresponding to the tag is added, and IP is carried out.Subsequently, a precipitated fraction is analyzed through a knownwestern blot technique. The tag-corresponding antibody may be bound to acarrier (e.g., agarose beads) in advance.

In this case, when nicastrin and the peptide formed of the entirety or aportion of Notch receptor or APP (including the intramembrane sequence)are precipitated in smaller amounts, the test antibody is determined tohave higher percent inhibition of γ-secretase activity.

Alternatively, binding assay may be carried out by immobilizing one ofnicastrin and the peptide on, for example, a carrier or an assay plate,and labeling the other with, for example, a radioisotope or afluorescent substance. A test antibody detected may be provided with atag (e.g., an antigen) or a label (e.g., a radioisotope).

Whether or not a test antibody inhibits γ-secretase activity may bedetermined through, for example, a method employing a GAL4-UAS systemand ELISA or a reporter gene in combination. In this case, a construct(SC100G) is prepared by inserting GAL4 into C99 through a known method,and a reporter construct (UAS-luc) is prepared by inserting a UASsequence into an upstream region of the luciferase gene serving as areporter gene. These constructs are introduced into host cells through aknown technique such as lipofection. Cells constitutively expressingnicastrin are selected by use of, for example, an antibiotic-resistantmarker as appropriate.

The constitutively expressing cells are cultured at 37° C. for 24 hours,and then a test antibody is exposed to the cells, followed by expressionof the transgene. Expression of the gene is induced by addition of 10 mMn-butylic acid. After culturing at 37° C. for 12 hours, the cells or theresultant supernatant is recovered.

In the case where the cells are employed, when the amount ofluminescence generated by luciferase is reduced after lysis of thecells, the test antibody is determined to inhibit binding betweennicastrin and a Notch receptor intramembrane peptide or an APP peptide,and to inhibit cleavage of the Notch receptor intramembrane peptide orthe APP peptide; i.e., the test antibody is determined to have highpercent inhibition of γ-secretase activity.

In the case where the supernatant is employed, an extracellularlyreleased Aβ peptide fraction having an indicator applicable to ELISA isassayed through ELISA. When the degree of ELISA reaction is low, thetest antibody is determined to inhibit binding between nicastrin andNotch receptor or an APP peptide, and to inhibit cleavage of theintramembrane sequence of a Notch receptor peptide or the APP peptide;i.e., the test antibody is determined to have high percent inhibition ofγ-secretase activity.

Alkaline phosphatase or GFP may be employed in place of luciferase.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1 Culturing of Insect Cells

Insect cells (Spodoptera frugiperda, Sf9) were cultured at 27° C. by useof Grace's Insect Media Supplemented (Invitrogen) containing 10% fetalbovine serum (FBS, Sigma), penicillin (100 U/mL), and streptomycin (100μg/mL) (Invitrogen). When mass culture was carried out, 0.001% pluronicF-68 (Invitrogen) was added to the aforementioned medium placed in a 1-Lspinner flask.

Example 2 Preparation of Recombinant Virus

Human nicastrin cDNA cloned into pEF6-TOPO/V5-His (Invitrogen)(pEF6-NCT) (T. Tomita et al., FEBS Lett. 520 (2002) 117-121) wassubcloned into pBlueBac4.5 (Invitrogen) so that the V5-His tag derivedfrom the vector was provided on the C-terminal side, to thereby preparea human-nicastrin-containing construct (pBlueBac4.5-NCT). Recombinantvirus preparation was carried out according to a protocol attachedBac-N-Blue Transfection Kit (Invitrogen). Specifically, Sf9 cells weretransfected with Bac-N-Blue DNA and the above-prepared plasmid (4 μg),followed by purification through a plaque assay (several times), tothereby prepare a recombinant virus containing only a target gene. Afterpreparation of a high titer stock, a titer of the virus was determinedthrough a plaque assay.

Example 3 Confirmation of Expression of Nicastrin on BV

Expression of nicastrin (i.e., a single-transmembrane protein) on BV wasconfirmed by use of the above-prepared recombinant virus. Sf9 cells wereinfected with the recombinant virus at a multiplicity of infection (MOI)of 5, and the cells and BV were recovered after 12 hours, 24 hours, 48hours, or 72 hours initiation of infection, followed by confirmation ofexpression of nicastrin through immunoblotting by use of ananti-nicastrin N-terminal antibody (anti-NCT (N-19), SantaCruz) and ananti-His antibody. As a result, nicastrin was found to be sufficientlyexpressed in both a cell fraction and a BV fraction 48 hours afterinitiation of infection. This indicates that, similar to the case ofSREBP-2 (Y. Urano, et al., Biochem. Biophys. Res. Commun. 308 (2003)191-196), nicastrin is expressed on BV.

Example 4 Preparation of Anti-Nicastrin Antibody by Use of Budding Virus(BV)

Since a large amount of gp64, which is a virus-derived membrane proteinand exhibits high antigenicity, is expressed on BV, when a mouse isinfected with BV, an anti-gp64 antibody is strongly induced, anddifficulty is encountered in yielding an antibody to a target antigen.Therefore, gp64 transgenic mice, which were prepared so as to exhibitresistance to gp64, were employed as mice for immunization.

Sf9 cells (5×10⁸ cells/500 mL) were infected withhuman-nicastrin-expressing recombinant virus (NCT-BV) at an MOI of 5,and cultured for 48 hours, followed by recovery of a culturesupernatant. BV serving as an antigen was prepared from the culturesupernatant through ultracentrifugation, and then gp64 transgenic micewere immunized five times with the antigen.

Screening of antisera and a resultant hybridoma culture supernatants wascarried out through BV-ELISA by a customary method. There were addedNCT-BV employed during immunization (serving as an antigen forimmobilization), and SREBP+SCAP-BV prepared through coinfection ofSREBP-2 and SREBP cleavage-activating protein (SCAP) (Y. Urano, et al.,Biochem. Biophys. Res. Commun. 308 (2003) 191-196) or wild-BV containingno foreign gene (20.mu.g/mL in saline) (serving as a negative control)(50.mu.L/well). As a result, there were yielded many clones which do notrespond to wild-type BV or SREBP/SCAP-expressing BV but show positiveresponse to only nicastrin-expressing BV (FIG. 1). Similarly, there wereyielded a plurality of clones (e.g., PPMX0401 and PPMX0410 (deposited asFERM AP-20895)) which recognize nicastrin expressed on BV, as determinedthrough immunoblotting employing BV (FIG. 2).

Example 5 Cell Culture

COS-7 cells (cells derived from simian kidney), HeLa cells (cellsderived from human cervical cancer), A549 cells (cells derived fromhuman lung cancer), or NKO cells (fibroblasts derived from nicastrinknockout mouse: T. Li, et al., J. Neurosci. 23 (2003) 3272-3277) werecultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL)(Invitrogen) at 37° C. and 5% CO₂.

Example 6 Identification of Antibody by Use of BV and Forced ExpressionProduct

The culture supernatants of positive clones selected through BV-ELISAwere subjected to SDS-PAGE and immunoblotting by use of NCT-BV, Wild-BV,human wild-type nicastrin, and mutant forms of nicastrin in the presenceof a 1×SDS-PAGE sample buffer. An anti-nicastrin N-terminal antibody(N-19) was employed as a positive control. In transient expression byuse of animal cells, transfection into COS-7 cells was carried out byuse of DEAE-dextran, and cells were recovered 48 hours after initiationof transfection. pEF6-NCT was employed for human wild-type nicastrin.Mutant nicastrin constructs (Δ312 and Δ694) were prepared from pEF6-NCTthrough long PCR (T. Tomita, et al., FEES Lett. 520 (2002) 117-121).

As a result, all the tested antibodies were found to recognizeexogenously expressed human wild-type nicastrin. The anti-nicastrinN-terminal antibody (N-19) (i.e., a positive control) or an antibody tothe C-terminal-added V5 tag recognized both wild-type nicastrin andmutant forms of nicastrin. In contrast, almost all the above-preparedantibodies (clones) (e.g., PPMX0401 and PPMX0410) did not recognizenicastrin Δ312 (FIG. 3). This suggests that the epitope site of each ofthe above-prepared antibodies is present in the extracellular domain ofnicastrin.

Example 7 Preparation of Cells Constitutively Expressing Nicastrin

For the purpose of analysis of anti-nicastrin antibodies, NKO cells weretransfected with pEF6-NCT by use of LipofectAmine (Invitrogen), and thenNKO cells constitutively expressing human nicastrin (NKO/NCT cells) wereselected in a medium containing 10 μg/mL blasticidin.

Example 8 Deglycosylation of Nicastrin

As has been known, nicastrin has, in the sequence thereof, 20 potentialglycosylation sites and highly undergoes N-linked glycosylation (T.Tomita, et al., FEBS Lett. 520 (2002) 117-121; J. Y. Leem, et al., J.Biol. Chem. 277 (2002) 19236-19249; D. S. Yang, et al., J. Biol. Chem.277 (2002) 28135-28142; and W. T. Kimberly, et al., J. Biol. Chem. 277(2002) 35113-35117).

Nicastrin is classified, on the basis of the degree of glycosylation,into mature nicastrin (molecular weight: about 130 kDa) and immaturenicastrin (molecular weight: about 110 kDa). Active γ-secretase complexcontains only mature nicastrin. Among N-linked sugar chains,complex-type sugar chains are known to exhibit resistance toendoglycosidase H (Endo H) but to be cleaved by peptide: N-glycosidase F(PNGase). Therefore, through Endo H treatment, the molecular weight ofcomplex-type glycosylated mature nicastrin is reduced to about 115 kDa,whereas the molecular weight of immature nicastrin having nocomplex-type sugar chain is reduced to about 80 kDa. In contrast,through PNGase F treatment, the molecular weights of both the maturenicastrin and immature nicastrin are reduced to about 80 kDa (D. S.Yang, et al., J. Biol. Chem. 277 (2002) 28135-28142, and W. T. Kimberly,et al., J. Biol. Chem. 277 (2002) 35113-35117).

In Example 8, a deglycosylation experiment was carried out for a purposeof epitopic analysis of anti-nicastrin antibodies.

Firstly, NKO/NCT cells were washed with PBS and then suspended in anRIPA buffer (50 mM Tris-HCl at pH 7.5, 1% Triton X-100, 1% sodiumdeoxycholate, 0.1% SDS, 150 mM NaCl), followed by inversion mixing at 4°C. for eight hours for lysis. Nicastrin was immunoprecipitated (IP) fromthe resultant lysate fraction by use of an anti-nicastrin. C-terminalantibody (N1660, Sigma), and the thus-precipitated nicastrin fractionwas employed for the following analysis.

For Endo H or PNGase treatment, 200 mM citrate-NaOH (pH 5.8), 0.1% SDS,and 1% 2-mercaptoethanol were added to the nicastrin fraction, and themixture was boiled at 95° C. for five minutes. 500 mU/mL EndoglycosidaseH (Roche Applied Sciences) or 200 U/mL PNGase F (Roche Applied Sciences)was added to the mixture, and reaction was carried out at 37° C.overnight. Finally, a 5× sample buffer (¼ amount of the reactionmixture) was added to the reaction mixture, and the resultant mixturewas boiled at 95° C. for five minutes, whereby reaction was terminated.

For neuraminidase (sialidase) treatment, 50 mM Na-acetate (pH 5.2), 2 mMCaCl₂, and 0.5% 2-mercaptoethanol were added to the nicastrin fraction,and the mixture was boiled at 95° C. for five minutes. 500 mU/mLNeuraminidase (Roche Applied Sciences) was added to the mixture, andreaction was carried out at 37° C. overnight. Finally, a 5× samplebuffer (¼ amount of the reaction mixture) was added to the reactionmixture, and the resultant mixture was boiled at 95° C. for fiveminutes, whereby reaction was terminated.

Samples prepared through treatment with the aforementioneddeglycosylation enzymes were subjected to western blot analysis. As aresult, PPMX0401, PPMX0408, and PPMX0410 were found to exhibitcross-reactivity to deglycosylated nicastrin (FIG. 4). In FIG. 4, “O”represents Endo H-resistant nicastrin; “black dot” represents completelydeglycosylated nicastrin; and “Δ” represents neuraminidase-desialylatednicastrin.

These data (in particular, the fact that each of the above-preparedantibodies recognized nicastrin which had been completely deglycosylatedby PNGase F) suggest that the antibody binds to nicastrin by recognizinga peptide chain of the protein rather than a sugar chain thereof.

Example 9 Immunoprecipitation (IP) of Endogenous Nicastrin by Use ofAnti-Nicastrin Antibody

HeLa cells were suspended in a cell homogenization buffer (10%glycerol-containing HEPES buffer (10 mM HEPES pH 7.4, 150 mM NaClcomplete inhibitor cocktail (Roche Applied Sciences))) and homogenizedby means of a homogenizer, followed by centrifugation at 1,500×g for 10minutes. Subsequently, the resultant supernatant was centrifuged at100,000×g for one hour, and the precipitate was employed as a HeLa cellmembrane fraction. The cell membrane fraction was lysed in a 1%CHAPSO-containing HEPES buffer, to thereby yield a HeLa cell membranelysate fraction. After IP of nicastrin from the lysate fraction by useof each of the above-prepared anti-nicastrin monoclonal antibodies,western blot analysis was carried out by use of various antibodies.

As a result, the above-prepared antibodies were found to be classifiedinto two groups; i.e., antibodies which allow IP of only immaturenicastrin (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) (first group);and antibodies which allow IP of both immature nicastrin and maturenicastrin (PPMX0406, PPMX0408, and PPMX0410) (second group) (FIG. 5). Inthe case of the antibodies of the second group (PPMX0406, PPMX0408, andPPMX0410), presenilin, PEN-2, and APH-1aL, which are components of theγ-secretase complex, were coprecipitated, whereas in the case of theantibodies of the first group (PPMX0401, PPMX0402, PPMX0407, andPPMX0409), only APH-1aL was precipitated in a small amount. As has beenreported, immature nicastrin binds to APH-1 and forms a sub-complexbefore it forms a γ-secretase complex (M. LaVoie, et al., J. Biol. Chem.278 (2003) 37213-37222). Therefore, conceivably, each of the antibodiesof the first group (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) bindsspecifically to immature nicastrin contained in a nicastrin-APH-1sub-complex.

As has also been reported, the structure of the extracellular domain ofnicastrin changes with formation of the γ-secretase complex (K.Shirotani, et al., J. Biol. Chem. 278 (2003) 16474-16477). When the HeLacell 1% CHAPSO lysate was treated with trypsin, the extracellular domainof nicastrin exhibited resistance to trypsin. Therefore, conceivably,the extracellular domain of nicastrin maintains its structure in theγ-secretase complex in the presence of 1% CHAPSO (FIG. 6). Thus, thedata of the IP experiment suggest that the epitope site of each antibodyof the first group is masked through structural change of nicastrin,whereas the epitope site of each antibody of the second group may beexposed even after structural change of nicastrin.

Example 10 Immunostaining of Cultured Cells by Use of Anti-NicastrinMonoclonal Antibody

Biochemical studies have reported that active γ-secretase containingmature nicastrin is localized to lipid rafts (Urano Y., Hayashi I., IsooN., et al.: Association of active γ-secretase complex with lipid rafts.J. Lipid Res. 2005, 46: 904). In this Example, cultured cells wereimmunostained by use of the above-prepared antibodies, to therebyexamine intracellular localization of nicastrin recognized by theantibodies. Intracellular localization of nicastrin was examined by useof HeLa cells and NKO cells. Cells were bonded, at an appropriate celldensity, to a cover glass which had been coated with poly-D-lysine inadvance, and the cells were washed with PBS and then fixed with PBScontaining 4% paraformaldehyde. PBS containing 3% BSA was employed forblocking, and, in the case of permeation, Triton X-100 (finalconcentration: 0.1%) was further added. Each of the antibodies wasdiluted to an appropriate concentration with a blocking solution, andthe thus-diluted antibody was reacted with the cells (at roomtemperature for three hours, or at 4° C. overnight). An anti-mouse oranti-rabbit immunoglobulin antibody bound to Alexa 488 or 546 wasemployed as a secondary antibody.

As a result, in HeLa cells, a granular structure and the cell membranewere stained in the presence of PPMX0408, whereas such a structure wasnot stained in the presence of PPMX0401 (FIG. 7). In NKO cells, agranular structure was not stained in the presence of PPMX0408. However,when wild-type nicastrin was introduced into NKO cells, there wasobtained a stained image similar to that obtained in the case of HeLacells (FIG. 8). These data indicate that the granular structure stainedin the presence of PPMX0408 is derived from nicastrin.

Subsequently, in order to examine intracellular localization of thegranular structure, co-staining was carried out by use of PPMX0408 andantibodies to various marker proteins. As a result, localization of thegranular structure did not correspond to that of calnexin and giantin,which are marker proteins for endoplasmic reticulum and Golgi body,respectively (FIG. 9). In contrast, the results of staining in thepresence of cholera toxin subunit B (CTB), which is used for staining ofGM1 ganglioside present in lipid rafts, corresponded well to those ofstaining of the granular structure in the presence of PPMX0408. Theresults of staining in the presence of PPMX0401 did not correspond tothose of staining in the presence of CTB (FIG. 10). Correspondence oflocalization similar to that described above was observed even undernon-permeating conditions (i.e., no treatment with Triton X-100 duringblocking) (FIG. 10). These data suggest that PPMX0408 recognizes maturenicastrin which is localized to lipid rafts (including cell membrane).

Example 11 Neutralization of Human Active γ-Secretase Activity by Use ofAnti-Nicastrin Monoclonal Antibody

Since PPMX0408 or PPMX0410 binds to mature nicastrin contained in activeγ-secretase under the conditions where the γ-secretase complex ismaintained, these antibodies are considered to affect γ-secretaseactivity. Therefore, a microsomal fraction of HeLa cells was lysed with1% CHAPSO; each of the antibodies was added to an in vitro γ-secretaseassay system employing an artificial substrate; and γ-secretase activitywas determined on the basis of accumulation of de novo synthesized Aβ(Takasugi N., Tomita T., Hayashi I., Tsuruoka M., Niimura M., TakahashiY., Thinakaran G., Iwatsubo T.: The role of presenilin cofactors in theγ-secretase complex. Nature 2003, 422: 438; and Takahashi Y., HayashiI., Tominari Y., et al.: Sulindac sulfide is a non-competitiveγ-secretase inhibitor that preferentially reduces Aβ 42 generation. J.Biol. Chem. 2003, 278: 18664).

When PPMX0401 was added (final concentration: 10 μg/mL), γ-secretaseactivity was maintained at almost the same level as in the case wherePBS was added. In contrast, when PPMX0408 or PPMX0410 was added (finalconcentration: 10 μg/mL), γ-secretase activity was inhibited by about20%, as compared with the case where PBS was added (FIG. 11). Thissuggest that the antibodies which bind to mature nicastrin exhibitγ-secretase inhibitory activity.

Example 12 Effect of Anti-Nicastrin Monoclonal Antibody on Viability ofCancer Cell Lines

Firstly, in order to identify a cancer cell line exhibitingNotch-signaling-dependent survival, the effect of a γ-secretaseinhibitor DAPT (H F. Dovey, et al., J. Neurochem. 76 (2001) 173-181) onsurvival of HeLa cells or A549 cells was evaluated through the MTTmethod. HeLa cells or A549 cells (5×10³ cells) were inoculated onto a96-well multiplate and treated with DAPT (final concentration: 100 μM)for 72 hours. After the 72-hour treatment, MTT diluted with PBS wasadded to the plate so that the final MTT concentration was 500 μg/mL,followed by culturing at 37° C. for three to four hours. Thereafter,stop solution (10% SDS, 0.01 M HCl) was added to the plate fortermination of reaction, and the plate was allowed to stand still at 37°C. overnight, followed by dissolution of produced formazan. The formazansolution was uniformly mixed through pipetting, and absorbance wasmeasured at 550 nm, to thereby calculate cell viability. As a result,the viability of DAPT-treated A549 cells was significantly lower thanthat of DAPT-untreated A549 cells. In contrast, no significantdifference was observed in viability between DAPT-treated HeLa cells andDAPT-untreated HeLa cells (FIG. 12). Subsequently, in order to confirmthat this reduction in cell viability was attributed to inhibition ofγ-secretase activity, endogenous nicastrin of A549 cells was knockeddown through treatment with nicastrin-corresponding short interferenceRNA (siRNA), and change in cell viability was determined. As a result,in the case of treatment with nicastrin siRNA, cell viability wasreduced by about 20%, as compared with the case of treatment with siRNAhaving a random sequence (scramble) (FIG. 13). Under the nicastrin siRNAtreatment conditions, expression of endogenous nicastrin was completelyinhibited (FIG. 14). These data suggest that, unlike the case of HeLacells, survival of A549 cells requires γ-secretase activity.

Subsequently, the effect of the above-prepared antibodies on survival ofA549 cells was examined. Each of the antibodies was added to A549 cellsso that the final antibody concentration was 10 μg/mL, and, 96 hoursafter addition of the antibody, cell viability was determined throughthe MTT method. As a result, the viability of PPMX0410-treated A549cells was significantly lower than that of antibody-untreated A549 cellsor PPMX0401-treated A549 cells (FIG. 15). These data suggest that anantibody exhibiting γ-secretase inhibitory activity has an ability toinhibit proliferation of cancer cells exhibiting γ-secretase-dependentsurvival.

Example 13 Effect of Anti-Nicastrin Monoclonal Antibody on Proliferationof Leukemia Cell Lines

As has been reported, proliferation of cells of the following celllines: TALL-1, ALL-SIL, and DND-41—which are isolated and establishedfrom patients with T-cell acute lymphoblastic leukemia (T-ALL)—requiresNotch signaling (Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P.t., Silverman, L. B., Sanchez-Irizarry, C., Blacklow, S. C., Look, A. T.and Aster, J. C. (2004), Activating mutations of NOTCH1 in human T cellacute lymphoblastic leukemia. Science 306, 269-271). As has also beenreported, in TALL-1 cells, somatic mutation is not found in the Notch1gene, but in ALL-SIL cells or DND-41 cells, missense mutation occurs inthe region (HDN) involved in interaction between an extracellular domainof Notch1 and TMIC (transmembrane-intracellular domain of Notch), anddeletion (by mutation) occurs in the PEST region involved in degradationof NICD (Notch intracellular domain) (FIG. 16). Conceivably, mutation ofthe HDN region causes ligand-independent heterodimeric dissociation,shedding, and cleavage by γ-secretase, and deletion in the PEST regionincreases the stability of NICD, which induces abnormal activation ofNotch signaling, thereby causing T-ALL.

Firstly, there was examined the effect of treatment of TALL-1, ALL-SIL,or DND-41 cells with a γ-secretase inhibitor on metabolism of Notch1.Through western blot analysis by use of an antibody mN1A to theintracellular ankyrin repeat domain of Notch1 (Chemicon, Cat #MAB5352),a band considered to be attributed to Notch1 TMIC was observed in thecases of all these types of cells. In the case of ALL-SIL cells orDND-41 cells, a band considered to be attributed to NEXT (Notchextracellular truncation) was observed at a position slightly below theTMIC band, and also a somewhat unclear band considered to be attributedto NICD was observed at a position below the NEXT band (FIG. 17). In thecase of ALL-SIL cells or DND-41 cells, constitutive expression of NICDwas determined by an antibody Val1744 (Cell Signaling, Cat #2421)specific to the cleaved N-terminal of NICD, but in the case of TALL-1cells, expression of NICD was not observed. Subsequently, a γ-secretaseinhibitor YO (concentration: 10, 100, or 1,000 nM) was added to theculture supernatant of each type of cells, and the cells were recovered48 hours after addition of YO, followed by western blot analysis of theresultant lysate. As a result, in the case of YO treatment of ALL-SILcells or DND-41 cells, NICD was found to disappear, and TMIC and NEXTwere found to be accumulated (FIG. 17). These data suggest that Notchsignaling is constitutively activated in at least both ALL-SIL cells andDND-41 cells.

Subsequently, the effect of YO treatment on proliferation of these cellswas examined. Cells were inoculated onto a 96-well plate (5×10³cells/well) and cultured at 37° C. overnight. Then, a γ-secretaseinhibitor YO was added to the plate, followed by culturing for sevendays. Thereafter, percent cell proliferation was determined by use ofAlamar Blue (Serotec). Alamar Blue was added to the culture liquid in anamount of 1/10 that of the culture liquid, followed by culturing at 37°C. for four hours. Subsequently, the resultant culture supernatant wasrecovered. Fluorescence in the culture supernatant was measured by meansof a plate reader (excitation wavelength: 530 nm, fluorescencewavelength: 590 nm), and percent cell proliferation was calculated byuse of the following formula.

${{cell}\mspace{14mu}{proliferation}\mspace{14mu}(\%)} = {\frac{A_{590}}{{PC}_{590}} \times 100}$

In the above formula, “A590” represents the absorbance of a sample at590 nm, and “PC590” represents the absorbance of a positive controlgroup (treated with PBS or DMSO) at 590 nm. As a result, proliferationof TALL-1 cells or DND-41 cells was found to be inhibited through YOtreatment. Specifically, through treatment with 10 nM YO, proliferationof TALL-1 cells or DND-41 cells was inhibited by about 60% or about 50%,respectively, and, through treatment with 1,000 nM YO, proliferation ofTALL-1 cells or DND-41 cells was inhibited by about 80% (FIG. 18).Unexpectedly, virtually no inhibition of cell proliferation was observedin ALL-SIL cells, in which NICD was found to disappear through YOtreatment (as determined by western blot analysis). These data indicatethat TALL-1 cells or DND-41 cells exhibit γ-secretase activity-dependentproliferation.

The above-obtained data suggest that, among the examined T-ALL-derivedcells, at least DND-41 cells exhibit Notch signaling/γ-secretaseactivity-dependent proliferation. Therefore, the effect of PPMX0410(i.e., an anti-nicastrin antibody) on proliferation of DND-41 cells wasexamined. PPMX0410 or a mouse IgG fraction (concentration: 0.1, 1, 10,or 100 μg/mL) was added to a DND-41 cell culture supernatant, followedby culturing for seven days. Thereafter, percent cell proliferation wasdetermined by use of Alamar Blue. As a result, percent cellproliferation tended to slightly increase in anIgG-fraction-concentration-dependent manner, but tended to lower throughaddition of PPMX0410. Specifically, proliferation of DND-41 cells wasinhibited by about 60% through addition of 100 μg/mL PPMX0410 (FIG. 19).These data indicate that PPMX0410 inhibits Notch signaling/γ-secretaseactivity-dependent proliferation of T-ALL cells.

On the basis of these results, PPMX0410 was deposited with InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (Central 6th, Tsukuba Center, 1-1-1, Higashi,Tsukuba, Ibaraki, Japan, Postal Code 305-8566) (deposition date: Apr.21, 2006, accession number: FERM-AP 20895).

Example 14 Effect of Anti-Nicastrin Monoclonal Antibody in InhibitingBinding Between Nicastrin and Substrate

As has been reported, nicastrin may function as a substrate receptor inthe γ-secretase complex (Shah S., Lee S F., Tabuchi K., Hao Y H., Yu C.,LaPlant Q., Ball H., Dann C E 3rd, Sudhof T., Yu G.: Nicastrin functionsas a γ-secretase-substrate receptor. Cell 2005, 122: 435).

Therefore, there was examined a possibility that PPMX0410 exhibitsγ-secretase inhibitory activity by inhibiting interaction betweenγ-secretase and a substrate therefor.

Firstly, there was expressed, in Sf9 cells, nicastrin (having a VS-Histag sequence added at the carboxyl terminus) or Ni 00-FLAG (100 aminoacid residues (No. 1711 to No. 1809) of Notch receptor including theintramembrane sequence(NH2-MVKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) (SEQ ID NO: 15)) andhaving a FLAG-His tag (DYKDDDDKGSHJETHHHH) (SEQ ID NO: 16)) added at thecarboxyl terminus), Lee S F., Shah S., Li H., Yu C., Han W., Yu G.:Mammalian APH-i interacts with presenilin and nicastrin and is requiredfor intramembrane proteolysis of amyloid-3 precursor protein and Notch.J. Biol. Chem. 2002 277: 45013). Subsequently, a cell membrane fractionwas prepared through the method described above in Example 9.

The resultant cell fraction was lysed in a HEPES buffer containing 1%CHAPSO, to thereby yield a nicastrin fraction or an N100 fraction.

The nicastrin fraction was mixed with PPMX0401 or PPMX0410 diluted to anappropriate concentration with PBS, and reaction was carried out at 4°C. overnight. Thereafter, the N100 fraction was added to the reactionmixture, followed by inversion mixing for three hours. A 1%CHAPSO-containing HEPES buffer was employed during mixing of thenicastrin fraction with the antibody, and a 0.5% CHAPSO-containing HEPESbuffer was employed after addition of the N100 fraction.

Nicastrin and N100-FLAG were coprecipitated from the resultantnicastrin-N100 fraction mixture by use of anti-V5-antibody-boundV5-agarose beads (SIGMA) or anti-FLAG-antibody-bound M2-agarose beads(SIGMA), and the precipitated fraction was subjected to western blotanalysis by use of an anti-V5 antibody (FIG. 20).

In the case where a nicastrin fraction which had been denatured with0.1% SDS in advance was employed, the amount of nicastrin precipitatedby the M2-agarose beads (i.e., nicastrin bound to N100-FLAG) wasreduced, as compared with the case where a native nicastrin fraction wasemployed (comparison between lanes “D” and “N” in FIG. 20).

Thus, this experiment system was considered to be applicable todetection of structure-dependent binding of nicastrin to N100.

Under the aforementioned conditions, PPMX0401 or PPMX0410 was added, andthe amount of nicastrin precipitated by the M2-agarose beads wasmeasured, followed by comparison of the resultant measurement data. Inthe case where PPMX0410 was added at a concentration of 10 or 100 μg/mL,the amount of nicastrin precipitated was found to be considerablyreduced (FIG. 21). When the measurement data were normalized with theamount of nicastrin precipitated by the V5-agarose beads, PPMX0410 (atthe aforementioned concentrations) was found to inhibit binding betweennicastrin and N100-FLAG by about 60% (FIG. 21).

These data suggest that PPMX0410 inhibits γ-secretase activity byinhibiting binding between nicastrin and a substrate for the enzyme.Thus, these data suggest that an antibody exhibiting potent γ-secretaseinhibitory activity can be selected on the basis of inhibition ofbinding between nicastrin and a substrate for the enzyme.

Example 15 Inhibition of γ-Secretase Activity by Anti-Nicastrin Antibodyin Living Cells

An experiment was carried out by means of a GAL4-UAS system employingreporter cells, in order to determine whether or not PPMX0410—whichinhibits γ-secretase activity in an in vitro reaction system—alsoinhibits cleavage mediated by γ-secretase activity in living cells.

C99 is a fragment produced through cleavage of APP (amyloid precursorprotein) by BACE (β-site APP cleaving enzyme) and serves as a directsubstrate for γ-secretase.

A preproenkephalin-derived signal peptide was inserted into C99, andGAL4 (i.e., a yeast-derived transcription factor) was insertedimmediately downstream of the transmembrance domain of C99, to therebyprepare a construct (SC100G). The construct (SC100G) was subcloned intopcDNA3.1/Hygro vector (Invitrogen).

GAL4/VP16 was bound to NΔE (including N99), in which deletion occurs inthe extracellular domain of Notch receptor and which serves as a directsubstrate for γ-secretase in a ligand-independent manner, to therebyprepare a construct (NΔEGV, Taniguchi Y., Karlstrom H., Lundkvist J.,Mizutani T., Otaka A., Vestling M., Bernstein A., Donoviel D., LendahlU., Honjo T.: Notch receptor cleavage depends on but is not directlyexecuted by presenilins. Proc. Natl. Acad. Sci. U.S.A 2002, 99: 4014).The construct (NΔEGV) was subcloned into pcDNA3.1 vector(pcDNA3.1-NΔEGV).

A UAS sequence was inserted upstream of luciferase in pGL3(R2.2) vector(Promega), to thereby prepare a construct (UAS-luc), and the constructwas employed as a reporter construct. eGFP was subcloned into pcDNA3(Invitrogen), and the thus-prepared pcDNA3-eGFP was employed as acontrol vector for monitoring the number of cells. HEK293 cells weretransfected with pcDNA3.1-SC100G and UAS-luc, or transfected withpcDNA3.1-NΔEGV, UAS-luc, and pcDNA3-eGFP by use of Lipofectamine 2000(Invitrogen). Cells constitutively expressing nicastrin (HEK/SC100Gcells or HEK/NΔEGV cells) were selected by use of anantibiotic-resistant marker (Hygromycin (Wako Pure Chemical Industries,Ltd.) or G418 (CALBIOCHEM), respectively).

HEK/SC100G cells or HEK/NΔEGV cells were inoculated into a 48-wellmultiplate (2.5×10⁴ cells). After culturing at 37° C. for 24 hours, PBSor PPMX0410 diluted to an appropriate concentration with PBS was addedto the plate. Cells treated with DMSO or DAPT (final concentration: 10μM) (i.e., γ-secretase-activity-inhibiting control) were also provided.After culturing at 37° C. for 36 hours, n-butyric acid (finalconcentration: 10 mM) was added for induction of transgene expression.After culturing for 12 hours, cells and a culture supernatant wererecovered, and the amount of Aβ was determined through a reporter assayor ELISA.

The recovered cells were lysed in a lysis buffer (Promega), and theresultant lysate was subjected to the reporter assay. PicaGene (Toyo InkMfg. Co., Ltd.) was employed as a luminescent substrate. The amount ofluciferase luminescence was normalized by the concentration of protein(in the case of HEK/SC100G cells), or by the amount of eGFP luminescence(in the case of HEK/NΔEGV cells), to thereby yield relative light unit(RLU). The amount of Aβ secreted in the culture supernatant ofHEK/SC100G cells was determined through ELISA, and the thus-determinedAβ amount was normalized by the concentration of protein similar to thecase of normalization of the amount of luciferase luminescence.

As a result, PPMX0410 was found to inhibit, in a concentration-dependentmanner, reporter activity (FIG. 22A) and Aβ secretion (FIG. 22B) inHEK/SC100G cells, and reporter activity (FIG. 22C) in HEK/NΔEGV cells.Under the aforementioned conditions, DAPT (i.e., a γ-secretaseinhibitor) was found to inhibit reporter activity in both HEK/SC100Gcells and HEK/NΔEGV cells.

These data indicate that PPMX0410 also inhibits γ-secretase activity inliving cells and inhibits intramembrane protein cleavage in APP or Notchreceptor.

The method described in Example 14 can be employed for high throughputscreening. Therefore, the method is considered applicable to selectionof an antibody exhibiting potent γ-secretase inhibitory activity.

1. A method for treatment of Alzheimer's disease, comprisingadministering an anti-nicastrin antibody, which is produced by ahybridoma PPMX0410 deposited as FERM-AP20895, to a subject sufferingfrom Alzheimer's disease.